bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
1 Analysis of contributory gut microbiota and lauric acid against necrotic enteritis in
2 Clostridium perfringens and Eimeria side-by-side challenge model
3
4
5 Short title: The impact of Clostridium perfringens challenge on gut microbiota in chickens
6
7 Wen-Yuan Yang1, Yuejia Lee1, Hsinyi Lu1, Chung-Hsi Chou2, Chinling Wang1*
8
9 1 Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University,
10 P.O. Box 6100, Mississippi State, MS 39762, 2 Zoonoses Research Center and School of
11 Veterinary Medicine, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei City,
12 106, Taiwan
13
14 *Corresponding author: [email protected]
1 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
16 Abstract
17 Gut microbiota has been demonstrated to be involved in intestinal nutrition, defense, and
18 immunity, as well as participating in disease progression. This study was to investigate gut
19 microbiota changes in chickens challenged with netB-positive Clostridium perfringens strain 1
20 (CP1) and/or the predisposing Eimeria species (Eimeria). In addition, the effects of lauric acid, a
21 medium-chain fatty acid (MCFA), on NE reduction and modulation of microbiota were
22 evaluated. The results demonstrated that microbial communities in the jejunum were distinct
23 from those in the cecum, and the microbial community change was more significant in jejunum.
24 Challenge of CP1 in conjunction with Eimeria significantly reduced species diversity in jejunal
25 microbiota, but cecal microbiota remained stable. In the jejunum, CP1 challenge increased the
26 abundance of the genera of Clostridium sensu stricto 1, Escherichia Shigella, and Weissella, but
27 significantly decreased the population of Lactobacillus. Eimeria infection on its own was unable
28 to promote NE, demonstrating decrements of Clostridium sensu stricto 1 and Lactobacillus. Co-
29 infection with CP1 and Eimeria reproduced the majority of NE lesions with significant
30 increment of Clostridium sensu stricto 1 and reduction in Lactobacillus. The changes of these
31 two taxa increased the severity of NE lesions. Further analyses of metagenomeSeq, STAMP, and
32 LEfSe showed significant overgrowth of Clostridium sensu stricto 1 was associated with NE and
33 Eimeria infection than C. perfringens challenge alone. The supplementation of lauric acid did
34 not reduce NE incidence and severity but decreased the relative abundance of Escherichia
35 Shigella. In conclusion, significant overgrowth of Clostridium sensu stricto 1 in the jejunm is the
36 major microbiota contributory to NE. Controlling proliferation of this taxon in the jejunum
37 should be the niche for developing effective strategies against NE.
2 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
38 Keywords: necrotic enteritis, Clostridium perfringens, Eimeria, lauric acid, microbiota
3 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
39 Introduction
40 Necrotic enteritis (NE) as the result of proliferations of Clostridium perfringens (C. perfringens)
41 type A and their associated toxins in the small intestine of chickens is a devastating enteric
42 disease, characterized by sudden diarrhea, unexpected mortality, and mucosal necrosis [1, 2]. Up
43 to 37% of commercial broiler flocks is estimated to be affected by this disease, and it has
44 contributed to the losses of 6 billion dollars in the global poultry industry [3, 4]. In recent
45 decades, C. perfringens-associated NE in poultry has been well-controlled by in-feed
46 antimicrobial growth promoters (AGPs) [5]. However, the emergence of antibiotic-resistant
47 bacteria from animals and the potential threat of transmission to humans has led to bans on using
48 AGPs in many countries [6, 7]. Following the withdrawal of AGPs from poultry feed, NE has re-
49 emerged as a significant disease to the poultry industry [8-11].
50 Gut microbiota is one of the central defense components in the gastrointestinal tract against
51 enteric pathogens, which works by modulating host responses to limit the colonization of
52 pathogens [12]. Interactions between gut microbiota and the host could influence intestinal
53 morphology, physiology, and immunity [13]. Recently, gut microbiota has been demonstrated to
54 regulate intestinal gene expression [14] and T cell-mediated immunity [15] as well as to
55 accelerate the maturation of the gut immune system [16]. Conversely, a growing number of
56 studies have observed gut microbial shifts in enteric diseases, considering that gut microbiota
57 plays a role in the progress of disease development. Similar results were also represented in NE
58 induction models, proposing that the disturbance of gut microbiota interacts with the host,
59 subsequently promoting the development of NE [17-20]. In the case of human necrotizing
60 enterocolitis, an enteric disease in infants associated with C. perfringens [21], a recent study
61 found that Bacteroides dorei, an opportunist pathogenic bacterium in anaerobic infections, was
4 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
62 associated with an increased mortality of this disease [22]. Furthermore, several studies have
63 demonstrated that the increment of bacteria belong to a genus of Escherichia-Shigella was
64 associated with C. perfringens infection [5, 20]. This evidence raised the possibility that certain
65 microbes or microbiota in the gut may contribute to the virulence or development of enteric
66 disease in chickens, particularly for NE.
67 The removal of AGPs drove the poultry industry to search for an alternative in prevention to
68 decrease the incidence of NE. Probiotics, prebiotics, organic acids, plant extracts, essential oils,
69 and enzymes arose in response to this demand, but the efficacy of those on NE reduction were
70 variable and inconsistent [23, 24]. However, a medium-chain fatty acid (MCFA), lauric acid, was
71 found to have strong in vitro antimicrobial activity against gram-positive organisms [25-27] and
72 C. perfringens [28, 29]. In an in vivo trial, lauric acid with butyric acid demonstrated the lowest
73 incidence and severity of NE compared to other treatments [29]. However, this promising result
74 did not promote more applications of lauric acid against NE, and the interaction of lauric acid
75 with gut microbiota was not even addressed. The evaluation of its modulation effect on NE
76 reduction and gut microbiota simultaneously would be valuable in exploring specific microbial
77 community contributory to NE.
78 Although C. perfringens is the causative etiological agent of NE, it is evident that other
79 predisposing factors are required for NE induction [10, 30-32]. Even though gut microbiota has
80 been suggested to be involved in the progress of NE development [33, 34], association between
81 microbiota profile and NE development have not been well elucidated. Most studies intensively
82 focused on changes of microbial communities in the ileum or cecum where higher quantity of
83 microbes or/and more diverse microbial compositions were harbored; however, most results
84 were inconclusive [17-19, 35, 36]. Inversely, microbiota in the jejunum, which serve as the
5 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
85 primary site for colonization of C. perfringens and development of NE [33], was seldom
86 evaluated. In the present study, we investigated gut microbiota targeting NE cases and in
87 chickens with side-by-side treatments with the causative pathogen and parasitic predisposing
88 factor, Eimeria, and expected to unveil the contributory microbe or microbiota to NE. The
89 effects of lauric acid on NE reduction and modulation of microbiota were also examined to
90 confer the alternative intervention to prevent and control NE.
91
92 Materials and methods
93 Ethics statement
94 All procedures for the care, housing and treatment of chickens were approved by the Institutional
95 Animal Care and Use Committee at Mississippi State University (IACUC 16-439).
96
97 Chicken, diet, and experimental design
98 A total of 50 male and female one-day-old unvaccinated broiler chicks (Cobb strain) were
99 obtained from a commercial hatchery. Chicks were inspected on receiving to ensure their healthy
100 status and randomly allotted to 5 groups, studying the NE incidence and gut microbiota after
101 challenge of netB-positive C. perfringens (A group: CP1), co-infection with netB-positive C.
102 perfringens and multi-species Eimeria (B group: CP1+Eimeria), addition of lauric acid to feed
103 chickens co-infected with netB-positive C. perfringens and multi-species Eimeria (C group:
104 CP1+Eimeria+LA), inoculation of multi-species Eimeria (D group: Eimeria), and no treatment
105 (E group: CTL).
106 Chicks in groups were placed in separate temperature-controlled iron tanks with nets in the
107 floor-pen facility and lined with fresh litter. Throughout the 19-day study period, wheat-based
6 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
108 diets prepared based on the formula by Branton et al. [37] were offered for the first 7 days, and
109 then the rations were replaced by fishmeal diets (wheat-based diets containing 50% fishmeal)
110 obtained from the 1:1 mixture of wheat-based diet with fishmeal 60 N (Seven Springs Farm,
111 Check, Virginia, USA), containing minimal 60% crude protein from days 8 until the end of the
112 study. For the lauric acid supplementing group, 400 mg of lauric acid powder (Fisher Scientific,
113 Pittsburgh, Pennsylvania, USA) was added into 1 kg of wheat-based diet or fishmeal diet to form
114 the final ration for chickens from day 8 onward [29].
115 Co-infection with netB-positive C. perfringens (CP1) and multi-species Eimeria was applied
116 to induce NE according to our previous studies. The success of reproducing NE was determined
117 by clinical signs and intestinal lesion scores reaching 2 or more. In brief, chickens in the co-
118 infection group were given a single gavage of coccidial inoculum at day 10, followed by oral
119 administration of 3 ml CP1 inoculum with average 2.5x108 colony-forming units (CFU)/ml at
120 day 15 for 4 consecutive days with a frequency of 3 times daily. For a single challenge of CP1 or
121 Eimeria, the same methodology and time points were conducted as in the co-infection group. All
122 chickens were inspected on a daily basis and humanely euthanized at day 19 by carbon dioxide.
123 Dead chickens not resulting from NE were excluded from the trial after necropsy. The
124 experimental trial was reviewed and approved by the Mississippi State University Institutional
125 Animal Care and Use Committee.
126
127 Challenge strain and inoculum preparation
128 Anticoccidial live vaccine containing live oocysts of E. acervulina, E. maxima, E. maxima MFP,
129 E. mivati, and E. tenella was used as a disposing factor. .The vaccine bottle contained 10,000
130 doses of oocysts in an unspecified proportion of Eimeria species. A ten-fold dose of vaccine was
7 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
131 prepared then applied on Eimeria-treated and co-infection groups. C. perfringens, a clinical NE
132 strain designated as CP1 obtained from Dr. John F. Prescott (Ontario Agricultural College,
133 University of Guelph, Canada), was used to challenge chickens. This strain was characterized as
134 netB-positive Type A and used to reproduce NE in a number of experiments [38-41]. CP1 was
135 cultured on blood agar plates and incubated anaerobically at 37°C for overnight. A single colony
136 was in turn transferred into 3 ml of fluid thioglycollate (FTG) medium (Himedia, Mumbai,
137 Maharashtra, India) at 37°C for overnight. Thereafter, the bacterial suspension was inoculated
138 into fresh FTG broth at a ratio of 1:10 and incubated at 37°C for 15, 19, and 23 hours,
139 respectively. The whole broth cultures were used to induce NE based on the evidence that
140 clostridia with toxins produce more severe disease than using cells alone [38]. The bacterial
141 concentration (CFU/ml) of inoculum was calculated by plate counting using Brain Heart Infusion
142 agar (Sigma-Aldrich, St. Louis, Missouri, USA), followed by anaerobic incubation at 37°C for
143 16 hours.
144
145 Sample collection and lesion scoring
146 Three chickens per group were randomly selected to collect fecal contents from the jejunum (AJ,
147 BJ, CJ, DJ, and EJ) and cecum (AC, BC, CC, DC, and EC). Among three CP1-challenged groups
148 (A, B, and C), chickens suffering NE (lesion score ≥ 2) were preferentially collected. Then, the
149 remaining chickens were sampled randomly to reach a quantity of 3. One percent of 2-
150 mercaptoethanol (Sigma-Aldrich) in PBS was used to wash fecal contents, and samples were
151 immediately frozen at -80°C. The intestinal tissues (duodenum to ileum) were inspected for NE
152 lesions and scored following the criteria described by Keyburn [42], with a range of 0 (no gross
153 lesions), 1 (congested intestinal mucosa), 2 (small focal necrosis or ulceration; one to five foci),
8 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
154 3 (focal necrosis or ulceration; 6 to 15 foci), and 4 (focal necrosis or ulceration; 16 or more foci).
155 Chickens with lesion scores reaching 2 or higher were identified as NE cases, and the highest
156 score in their small intestinal sections (duodenum, jejunum, and ileum) was recorded as the final
157 score of NE.
158
159 DNA extraction
160 Total genomic DNA was isolated from approximately 250 mg of fecal contents using the
161 MOBIO PowerFecal® DNA Isolation Kit (Mobio, Germantown, Maryland, USA) following the
162 manufacturer’s protocol with some modifications. After adding bead solution and lysis buffer,
163 the mixture was heated in a water bath at 65°C for 30 minutes followed by 5 minutes of
164 vortexing. The concentration and quality of harvested DNA were determined by NanoDrop™
165 One Microvolume UV-Vis Spectrophotometer (Fisher Scientific) and visualized on 0.8%
166 agarose gel (BD Biosciences, San Jose, California, USA). Afterward, genomic DNA was stored
167 at -20°C until further analysis.
168
169 16S rRNA library preparation and sequencing
170 The variable V3-V4 region of the 16S rRNA gene was PCR-amplified in 25-μl reaction mixtures,
171 containing 12.5 μl Clontech Labs 3P CLONEAMP HIFI PCR PREMIX (Fisher Scientific), 1 μl
172 of each 10-μm Illumina primer (forward primer-5’CCTACGGGNGGCWGCAG 3’ and reverse
173 primer-5’ GACTACHVGGGTATCTAATCC 3’) with standard adapter sequences, and 1 μl of
174 DNA template. The PCR conditions started with an initial denaturation step at 95°C for 3
175 minutes, followed by 25 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30
176 seconds, and a final extension step at 72°C for 5 minutes on Applied Biosystems GeneAmp PCR
9 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
177 System 9700 (Applied Biosystems Inc., Foster City, California, USA). The amplicons were
178 cleaned up by Monarch® DNA Gel Extraction Kit (New England Biolabs, Ipswich,
179 Massachusetts, USA). Subsequently, an index PCR was performed by using Nextera XT Index
180 Kit (Illumina, San Diego, California, USA) to attach a unique 8-bp barcode sequence to the
181 adapters. The applied 25-μl reaction was composed of 12.5 μl KAPA HiFi HotStart Ready Mix
182 (Kapa Biosystems, Wilmington, Massachusetts, USA), 2.5μl of each index primer, and 1 μl of
183 16S rRNA amplicon and reaction conditions were as follows: 95°C for 3 minutes, 8 cycles of
184 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and 72°C for 5 minutes on
185 Mastercycler® pro (Eppendorf AG, Hamburg, Germany). The PCR products were purified using
186 Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, Indiana, USA), and the size and
187 concentration were determined by Bioanalyzer with DNA 1000 chip (Agilent, Santa Clara,
188 California, USA) and Qubit® 2.0 Fluorometer with Qubit™ dsDNA HS Assay Kit (Fisher
189 Scientific). Those libraries were normalized and pooled to one tube with the final concentration
190 of 10 pM. Samples were thereafter sequenced on the MiSeq® System using Illumina MiSeq
191 Reagent Kit v3 (2×300 bp paired-end run).
192
193 Sequence processing and data analysis
194 Paired-end sequences were merged by means of fast length adjustment of short reads (FLASH)
195 v1.2.11 [43] after trimming of primer and adapter sequences. Reads were de-multiplexed and
196 filtered by Quantitative Insights into Microbial Ecology (Qiime) software v1.9.1 [44], meeting
197 the default quality criteria and a threshold phred quality score of Q ≥ 20. Chimeric sequences
198 were filtered out using the UCHIME algorithm [45]. The pick-up of operational taxonomic units
199 (OTUs) was performed at 97% similarity by the UPARSE algorithm [46] in USEARCH [47].
10 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
200 The OTUs were further subjected to the taxonomy-based analysis by RDP Classifier v2.11 with
201 a cut-off of 80% [48] using the Silva v128 database. Differential abundance of OTU among
202 treatments was evaluated by metagenomeSeq. The clustered OTUs and taxa information were
203 used for diversity and statistical analyses by Qiime v1.9.1 and R package v.3.3.1 (http://www.R-
204 project.org/). Differences of taxonomic profiles between groups were compared using Statistical
205 Analysis Metagenomic Profiles (STAMP) software [49] v2.1.3 with Welch’s t-test.
206 Furthermore, LEfSe (linear discriminant analysis effect size) from the LEfSe tool
207 (http://huttenhower.sph.harvard.edu/lefse/), an algorithm for high-dimensional class comparisons
208 between biological conditions, was used to determine the significant feature taxa between groups
209 or intestinal location. It emphasizes statistical significance, biological consistency, and effect
210 relevance and allows researchers to identify differentially abundant features that are also
211 consistent with biologically meaningful categories [50]. The Kruskal-Wallis rank sum test was
212 included in LEfSe analysis to detect significantly different abundances and performed LDA
213 scores to estimate the effect size (threshold: ≥ 4).
11 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
214 Results
215 NE reproduction and effects of lauric acid as an alternative prevention
216 Six of the NE cases were identified in three CP1-challenged groups (Table 1). They showed
217 different degrees of characteristic gross lesions in small intestinal tissues. The most severe
218 lesions were found in the jejunum, between its proximal end and Meckel’s diverticulum. Under
219 co-infection with CP1 and Eimeria, the incidence and severity of NE increased. No NE mortality
220 was noticed. Statistically significant differences of lesion score (LS) were determined between
221 three CP1-challenged groups (A, B, and C) and the control counterpart (p ≤ 0.05). The co-
222 infection groups (B and C) demonstrated a highly significant difference (p ≤ 0.01). However, the
223 supplementation of lauric acid did not reduce the incidence and severity which were similar to
224 the NE positive control group.
225
226 Table 1. NE frequency and mean lesion score by groups NE lesion score NE Group Treatment Subtotal Lesion score 0 1 2 3 4 case A CP1 0 9 1 0 0 10 1.11 ± 0.31a 1 B CP1+Eimeria 0 8 0 1 1 10 1.50 ± 1.02a 2 C CP1+Eimeria+LA 0 7 1 1 1 10 1.60 ± 1.02a 3 D Eimeria - - - - - 10 - 0 E CTL 5 4 0 0 0 9 0.44 ± 0.50b 0
227 LA lauric acid; NE necrotic enteritis; CTL: control group. 228 NE case: lesion score reaching 2 or above. 229 One chick in CTL was misclassified during the trial and excluded. 230 Dissimilar letters indicate a significant difference at a level of α=0.05.
231
232 Metadata and sequencing
12 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
233 A total of 11,191,102 sequence reads with an average length of 453 ±5 base pairs were obtained
234 from 30 samples, including 15 jejunal samples (3 samples per group in AJ, BJ, CJ, DJ, and EJ)
235 and 15 cecal samples (3 samples per group in AC, BC, CC, DC, and EC). The sequences were
236 filtered and further clustered into OTU using a cut-off of 97% similarity. The estimate of Good’s
237 coverage reached 98% for all the jejunal and cecal samples. The rarefaction curve demonstrated
238 that the sequencing depth was adequate to cover the bacterial diversity in the jejunal and cecal
239 samples (Figure S1).
240
13 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
241 Normal microbial composition in the jejunum and cecum
242 Firmicutes (92.1% of relative abundance) was the most dominant phylum in the jejunum,
243 followed by Cyanobacteria (2.2%) and Proteobacteria (2.1%), Bacteroidetes (1.9%), and
244 Actinobacteria (1.7%). On the contrary, the phylum of Bacteroidetes (75.5%) predominated in
245 the cecum, followed by Firmicutes (19.8%) and Proteobacteria (4.7%) (Figure 1A). At the
246 genus level, jejunal contents were dominated by Lactobacillus (41.2% of relative abundance) and
247 Clostridium sensu stricto 1 (39.1%), followed by other unclassified genus (8.7%), Weissella
248 (3.6%), Enterococcus (1.9%), Escherichia Shigella (1.8%), and Staphylococcus (1.6%).
249 Bacteroides (75.5%) was the most abundant genus in the cecum, followed by other unclassified
250 genus (17.2%), Escherichia Shigella (3.1%), Eisenbergiella (1.7%), and Anaerotruncus (1.5%)
251 (Figure 1B). The genera of Lactobacillus, Clostridium sensu stricto 1, Weissella, Enterococcus,
252 Staphylococcus, and Bifidobacterium in the jejunum exhibited significant difference in
253 abundance compared to those in the cecum. Cecal microbiota contained significantly higher
254 abundance of Bacteroides and Proteus (Welch’s t test, p < 0.05; Figure S2).
255 256
14 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
257 A)
258 259
15 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
260 (B)
261 262
16 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
263 (C)
264 Figure 1. Microbiota composition in jejunum and cecum with different treatments. Each bar
265 represents the average relative abundance of each bacterial taxon within a group. The top 5 and
266 10 abundant taxa are shown at the level of phylum and genus, respectively. (A) Abundant phyla
267 in jejunum and ceca by groups; (B) Abundant genera in jejunum and ceca by groups; (C)
268 Abundant genera in jejunal and cecal samples. LS stands for lesion score of NE.
17 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
269 Changes of microbial communities in response to treatments
270 In the jejunum, challenge of CP1 increased the relative abundance of the genera of Clostridium
271 sensu stricto 1 (54.75%), Escherichia Shigella (9.57%), and Weissella (4.99%) but significantly
272 decreased the population of Lactobacillus (25.44%) (Figure 2). The inoculation of Eimeria to
273 chickens significantly increased the relative abundance of Weissella (16.01%) and
274 Staphylococcus (6.51%), but decreased the amount of Lactobacillus (30.66%) and Clostridium
275 sensu stricto 1 (27.69%). Co-infection with CP1 and Eimeria led to significant increment of
276 Clostridium sensu stricto 1 (71.89%), increased relative abundance of Escherichia Shigella
277 (4.68%), but the decrements of Lactobacillus (16.99%), Weissella (0.44%) and Staphylococcus
278 (0.40%). In the cecum, different treatments did not promote significant difference of taxa
279 abundance between groups with an exception of Eisenbergiella, significantly increased in co-
280 infection group. However, challenge of CP1 and co-infection of CP1 and Eimeria still promoted
281 cecal increments of Clostridium sensu stricto 1 (the relative abundance of this taxon in groups
282 challenging with CP1, CP1 and Eimeria, and control was 0.75%, 1.99%, and 0.02%).
283
284
18 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
285 (A)
286 287 (B)
288
19 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
289 (C)
290 291
20 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
292 (D)
293 294
21 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
295 (E)
296 297
22 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
298 (F)
299 300 Figure 2. Differential abundance of genera between groups by metagenomeSeq. (A)
301 Lactobacillus; (B) Clostridium sensu stricto 1; (C) Weissella; (D) Staphylococcus; (E)
302 Escherichia Shigella; (F) Eisenbergiella. * p ≤ 0.05 and ** p ≤ 0.01.
303
304 Microbial diversities in response to treatments
305 In jejunal microbiota, challenge of CP1 (AJ) and co-infection with CP1 and Eimeria (BJ)
306 reduced species richness and evenness, but the infection of Eimeria (DJ) exerted counter results.
307 Addition of lauric acid into co-infection group (CJ) exacerbated the reduction observed in BJ
308 group. However, no apparent effect was noted on cecal microbiota following above treatments
309 (Figure S3 and Figure 3). Analysis of alpha diversity by Shannon index further demonstrated
310 that challenge of CP1 in conjunction with Eimeria infection significantly reduced species
311 diversity in jejunal microbiota. The 16S rRNA gene survey by principal coordinate analysis
23 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
312 (PCoA) and principal component analysis (PCA) showed a distinct separation of two community
313 profiles between the jejunal and cecal microbiota. Cluster and heat map analyses exhibited
314 distinct classifications and microbial compositions between the jejunum and cecum, coinciding
315 with observations on PCoA and PCA (Figure 4). Additionally, the results of PCoA and PCA
316 also depicted the differential diversity between the CP1-challenged (group AJ, BJ and CJ),
317 Eimeria-infected (DJ), and control (EJ) groups in jejunal microbiota, showing that challenge of
318 CP1 shared similar microbial community structures with co-infection with CP1 and Eimeria.
319 However, cecal groups with CP1 treatments did not display cluster phenomenon as jejunal
320 groups displayed in PCoA. PCA with hierarchical clustering further reflected that Clostridium
321 sensu stricto 1 was contributory to the similarity of NE assemblage, and the genera of
322 Lactobacillus, Weissella, and Staphylococcus contributed to discrepant community structures in
323 Eimeria-treated and control groups in the jejunum. On the other hand, Bacteroidetes was the
324 main genus contributing to the distinct separation between jejunal and cecal groups (Figure 5).
325
326
24 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
327 (A)
328 329
25 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
330 (B)
331 332
26 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
333 (C)
334 335
27 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
336 (D)
337 338 Figure 3. Comparison of microbial diversity between groups in jejunum and cecum using
339 different measures of alpha diversity. (A) Shannon index, (B) Simpson index (C), abundance-
340 based coverage estimator (ACE) index, and (D) Chao1 index. Results are shown as mean ±
341 SEM. Kruskal-Wallis test: * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.
342
28 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
343 (A)
344 345 (B)
346 347
29 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
348 (C)
349 350 (D)
351 352 Figure 4. Comparison of the compositions and similarities of jejunal and cecal microbiota with
353 different treatments. (A) Weighted Unifrac principal coordinate analysis (PCoA); (B) principal
354 component analysis (PCA); (C) cluster analysis by unweighted paired-group method using
30 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
355 arithmetic means (UPGMA) using unweighted Unifrac distance; (D) heat map analysis at the
356 genus level.
357
358 359 Figure 5. Principal component analysis and hierarchical clustering of contributory genus to NE
360 assemblage (in red circle) and to the dissimilarity between groups.
361
362 Microbial community structure and taxa contributory to NE
363 Analysis of jejunal microbiota in NE cases revealed that Clostridium sensu stricto 1, to which
364 causative C. perfringens belongs, was the most dominant genus, followed by Lactobacillus,
365 Weissella, Escherichia Shigella, Staphylococcus, and others. Accompanying the elevation of NE
366 severity, the relative abundance of Clostridium sensu stricto 1 increased (relative abundance ≥
367 75% in LS4 compared to 50-75% in LS2 and LS3). Conversely, the population of Lactobacillus
368 decreased while the lesion score was elevated. The relative amount of Escherichia Shigella was
31 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
369 variable in NE cases, presenting higher abundance after CP1 challenge but low population
370 following co-infection with CP1 and Eimeria. (Figure 1C).
371 Heat map analysis exhibited that NE cases harbored the similar microbial community profile.
372 Clostridium sensu stricto 1 and C. perfringens were consistently presented and abundant taxa in
373 jejunum (Figure 6). Opposite low abundance of Lactobacillus was noted. However, only the
374 increment of Clostridium sensu stricto 1 but not C. perfringens (data not shown) demonstrated
375 significance on NE by metagenomeSeq (Figure 2B). Using Welch's t-test, jejunal groups further
376 showed that CP1 in conjunction with Eimeria increased significantly Clostridium sensu stricto 1
377 and C. perfringens when compared to the control (Figure S4; p < 0.05), whereas challenge of
378 CP1 alone did not lead to significant increase of these taxa. Differential abundant phylotypes
379 between different treatments in jejunum were further evaluated by LEfSe using the LDA score of
380 4. This threshold guarantees that the meaningful taxa is compared and eliminates most of rare
381 taxa. LEfSe demonstrated similar results as Welch’s test that challenge of CP1 unable to yield a
382 significantly higher amount of Clostridium sensu stricto 1 and C. perfringens. However,
383 significant differences were displayed when CP1 co-infected with Eimeria (Figure 7). No
384 differential taxon was found in cecal groups (AC, BC, CC, HDC, and EC) while Welch's t-test
385 and LEfSe were applied.
386
387
32 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
388 (A)
389 390
33 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
391 (B)
392 393
34 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
394 (C)
395
396 397 Figure 6. Heat map analysis of contributory taxa to NE at the genus level in jejunal (A) and
398 cecal (B) samples. (C) Heat map of gut bacteria with the relative abundance of OTUS by z score
35 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
399 and represented bacterial taxa information, including phylum, family, genus, and species. Top 26
400 taxa was shown.
401
402 (A)
403 404
36 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
405 (B)
406 407 Figure 7. LEfSe identified the most differentially abundant clades at all taxonomic levels
408 between jejunal groups using the LDA score of 4. Differentially abundant taxa in group BJ
409 versus EJ (A) and CJ versus EJ (B).
410
411 Comparison of gut metagenomes in co-infected chickens with and without lauric acid
412 Addition of lauric acid increased the relative abundance of Clostridium sensu stricto 1 and
413 Weissella but decreased the relative amount of Escherichia Shigella in the jejunum compared to
414 the co-infection group without supplementing lauric acid. Nonetheless, no significance was
415 detected in this comparison. In addition, supplementation of lauric acid did not apparently affect
416 the cecal microbiota between these two groups.
37 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
417 Discussion
418 By exploring microbial composition in normal chickens, the major microbial genera in the
419 jejunum were Lactobacillus and Clostridium sensu stricto 1, followed by other unclassified
420 bacteria, Weissella, Enterococcus, Escherichia Shigella, and Staphylococcus. Bacteroides was
421 the most abundant group in the cecum, and the remaining taxa were sequentially other
422 unclassified bacteria, Escherichia Shigella, Eisenbergiella, and Anaerotruncus. Side by side
423 treatments of C. perfringens and Eimeria altered microbial community compositions,
424 significantly in jejunal microbiota. In this study, challenge of CP1 increased the abundance of
425 Clostridium sensu stricto 1, Escherichia Shigella, and Weissella in the jejunum, but significantly
426 decreased the population of Lactobacillus. Infection of Eimeria significantly increased the
427 abundance of Weissella and Staphylococcus, but decreased the amount of Lactobacillus and
428 Clostridium sensu stricto 1. Co-infection with C. perfringens and Eimeria led to significant
429 increment of Clostridium sensu stricto 1, increased abundance of Escherichia Shigella, but
430 decrements of Lactobacillus, Weissella and Staphylococcus. Specifically, it decreases the α-
431 diversity index of the small intestinal microbial community, promoting single dominance of
432 Clostridium sensu stricto 1 reaching the relative abundance to 71.89%. On the other hand, six
433 NE cases shared similar microbial community profile observed in PCA, indicating there exists a
434 certain microbiota contributory to the disease. With more NE severity, higher relative abundance
435 of Clostridium sensu stricto 1 but lower relative amount of Lactobacillus in jejunal microbiota
436 was noted.
437 Several studies has been shown C. perfringens challenge decreased the population of
438 Lactobacillus in ileum [20, 51]. Lactobacilli are known as lactic acid producing bacteria and
439 shown to have protection at intestinal barrier by competition with pathogens. They are also able
38 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
440 to induce immunomodulation and ferment carbohydrates into lactic acids that lower the pH of
441 the intestinal environment to inhibit growth of acid-sensitive pathogenic bacteria [52, 53].
442 Therefore, suppression of lactobacilli is regularly considered beneficial to growth and
443 colonization of enteric pathogen. This study first demonstrated the decrement of lactobacilli in
444 jejunum following challenge of C. perfringens alone and in conjunction with Eimeria. The
445 change of this taxon following the NE severity indicates that decrement of Lactobacillus may
446 play a role in the development of NE. In addition, the increased abundance of Escherichia
447 Shigella was also observed after the challenge of C. perfringens and co-infection with C.
448 perfringens and Eimeria. This genus includes enteric pathogens, which can colonize in the
449 intestines of both humans and chickens, consequently triggering specific diseases [54]. Some
450 studies indicated that the increment of Escherichia Shigella in ileum was correlate with NE [55,
451 56]. Nevertheless, our study found that C. perfringens challenge could increase the abundance of
452 Escherichia Shigella but the increment was not in accordance with NE occurrence. Furthermore,
453 the reduction of this taxa abundance was noticed in lauric acid supplementing group which has
454 higher number of NE cases. Those finding reflected a contradiction for this genus participating in
455 NE development. Last but not least, a reduced abundance of Weissella in the jejunum of NE
456 afflicted chickens was also noted. Another study reported similar result in cecal micorbiota after
457 C. perfringens challenge [18]. Weissella are lactic acid bacteria and belong to the family of the
458 Leuconostocaceae. They harbor probiotic properties and can generate several products with
459 prebiotic potential [57]. It may interact with C. perfringens as other lactic acid bacteria, but its
460 role in NE development is unclear. More studies will be needed to elucidate the relationship
461 between Weissella and NE.
39 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
462 In current study, significant overgrowth of Clostridium sensu stricto 1 was associated with NE
463 and the infection of Eimeria precedent to challenge of C. perfringens exerted synergistic effects
464 on the overrepresentation. This correlation was consistently demonstrated by analyses of
465 metagenomeSeq, STAMP, and LEfSe. The STAMP and LEfSe further showed C. perfringens
466 was significantly overrepresented in NE groups. However, such significance was not identified
467 by metagenomeSeq when C. perfringens was targeted. This result indicates that, in addition to C.
468 perfringens, other bacteria under the same genus of Clostridium sensu stricto 1 also played a role
469 in contributing to the development of disease. The Clostridium genus is well-classified into 19
470 clusters by phylogenetic analysis [58]. Clostridium sensu stricto are grouped around the type
471 species Clostridium butyricum and belong to the Clostridium cluster 1within the Clostridiaceae
472 family [59]. Clostridium sensu stricto 1 contains C. perfringens and other real Clostridium
473 species. Their members are generally perceived as pathogenic [60] as well as interpreted as an
474 indicator of a less healthy microbiota [61]. This suggestion coincides with our finding that C.
475 perfringens challenge on its own is not capable of causing significant abundance of Clostridium
476 sensu stricto 1 and unable to produce more NE case observed. Future research is recommended
477 to clarify the role of other members of Clostridium sensu stricto 1 in the pathogenesis of NE.
478 Single infection of Eimeria could not produce NE in the present study. The treatment reduced
479 the relative abundance of Clostridium sensu stricto 1 and Lactobacillus, but significantly
480 increased Weissella and Staphylococcus in jejunal microbiota. Eimeria infection has been shown
481 to provide nutrients for C. perfringens to grow and cause physical damage to gut epithelium, thus
482 facilitating the colonization and proliferation of C. perfringens [8, 62, 63]. However, the
483 inoculation of Eimeria into normal chickens did not elicit overgrowth of Clostridium sensu
484 stricto 1 and C. perfringens except challenging with exogenous C. perfringens. In contrast,
40 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
485 challenge of C. perfringens alone and in conjunction with Eimeria both promote proliferation of
486 Clostridium sensu stricto 1 and NE case. This indicates that the amount of commensal C.
487 perfringens in the jejunum under Eimeria infection is not sufficient to reach the significant
488 abundance of Clostridium sensu stricto 1 or C. perfringens, subsequently promoting the
489 occurrence of NE. Therefore, it is reasonable to suggest that the quantity of Clostridium sensu
490 stricto 1 or C. perfringens in jejunum is critical for the onset of proliferation. A recent study used
491 commensal C. perfringens, the isolate from normal chicken, to challenge broiler and reproduce
492 NE in conjunction with infection of E. maxima [64]. This result also highlighted that not the
493 specific C. perfringens strain but the exogenous addition of C. perfringens played the key in
494 achieving the consequence. Accordingly, the methodology to inhibit overgrowth of Clostridium
495 sensu stricto 1 or C. perfringens in small intestines will be the straightforward strategy to prevent
496 NE.
497 Recent studies have been shown that cecal microbiota had a prominent role in feed efficiency [65]
498 and received increasing attention in terms of diseases [66] and metabolism [67]. In this study, the
499 result of PCoA and PCA demonstrated that microbial communities in the jejunum were different
500 from those in the cecum. Side by side treatments of C. perfringens and Eimeria promoted
501 microbial shifts with biological significance in the jejunum but minimal fluctuations in taxa
502 abundance in the cecum. Comparatively, jejunal microbiota was more significant than cecal
503 microbiota to address characteristic gut microbiota contributory to NE by means of
504 metagenomeSeq and LEfSe analysis. The reason might be that cecal microbiota is demonstrated
505 more diverse than other intestinal sections [68] and inhibits higher amounts of microbes (1010-
506 1011 CFU/g) than those in the jejunum (108-109 CFU/g) [69]. Those may provide the buffer
507 effect on microbial changes in cecal microbiota. Besides, preferential colonization of C.
41 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
508 perfringens on mucosa of the small intestine [33] may also contribute to less amount of C.
509 perfringens into cecum, hence adverse to elicit significant changes in cecal microbiota.
510 Medium-chain fatty acids (MCFAs) such as lauric acid are a family of saturated 6- to 12-
511 carbon fatty acids from plants and documented beneficial effects on intestinal health and
512 microbial growth inhibition [70-72]. The mechanism for their bactericidal activity is not fully
513 understood. Relative studies showed that they could act as nonionic surfactants to become
514 incorporated into the bacterial cell membrane, as well as diffuse through cell membranes and
515 create pores, changing membrane permeability and leading to cell death [73-75]. In this work,
516 lauric acid attracted interest due to its inexpensiveness and natural properties, including strong
517 antibacterial effects against C. perfringens and no inhibitory effect on Eimeria infection [76].
518 Based on Timbermont’s study, lauric acid was most effective in inhibiting the growth of C.
519 perfringens strain in vitro. Given a supplementary dose of 0.4 kg/ton in feed caused a significant
520 decrease in NE incidence (from 50% down to 25%) compared with the infected, untreated
521 control group [29]. This study followed the dose and used experimental grade product of lauric
522 acid to evaluate the effects on NE incidence and intestinal microbiota. However, the addition of
523 lauric acid did not reduce the incidence of NE. For intestinal microbiota, lauric acid neither
524 exerted the inhibitory effect against proliferation of C. perfringens nor elevated the level of
525 beneficial bacteria, such as Lactobacillus and Bifidobacterium. But, the relative abundance of
526 Escherichia Shigella was decreased without affecting the incidence. Since lauric acid has
527 different grade of products, such as experimental or food grade, the contradictory result may
528 attribute to the influence of different formula on the absorptive efficiency of this compound.
529 MCFAs are hydrophobic and partly absorbed through the stomach mucosa. Hence, their
42 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
530 triacylglycerols are considered as a desirable formula for feed additive because they can be
531 absorbed intact into intestinal epithelial enterocytes via this form [77].
532 In summary, significant overgrowth of Clostridium sensu stricto 1 in jejunum was recognized
533 as the major microbiota contributory to NE. In addition to C. perfringens, other member within
534 Clostridium sensu stricto 1 was also found to participate in disease development. The decrement
535 of Lactobacillus following the NE severity indicated that lactobacilli also participate in the
536 progress of disease. These taxa showed counteractive effects in their functions as well as in the
537 bacterial abundance, attempting to maintain the homeostasis of jejunal microbiota in chickens.
538 Therefore, manipulations to inhibit multiplication of Clostridium sensu stricto 1 and C.
539 perfringens and to rehabilitate the dominant Lactobacillus population in the jejunum should be
540 the niche for developing effective strategies to prevent NE.
541
542 Acknowledgments
543 The authors would like to thank Dr. John F. Prescott (University of Guelph, Ontario, Canada) for
544 providing netB positive C. perfringens strain CP1. This work was supported by the USDA,
545 National Institute of Food and Agriculture (CRIS Project Accession Number 1014508) and the
546 College of Veterinary Medicine, Mississippi State University.
547
548 References
549 1. Cooper KK, Songer JG, Uzal FA. Diagnosing clostridial enteric disease in poultry. J Vet
550 Diagn Invest. 2013;25(3):314-27. doi: 10.1177/1040638713483468. PubMed PMID:
551 23572451.
43 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
552 2. Martin TG, Smyth JA. Prevalence of netB among some clinical isolates of Clostridium
553 perfringens from animals in the United States. Vet Microbiol. 2009;136(1-2):202-5. Epub
554 2008/12/17. doi: 10.1016/j.vetmic.2008.10.026. PubMed PMID: 19081686.
555 3. Van Der Sluis W. Clostridial enteritis is an often underestimated problem. World poultry.
556 2000;16(7):42-3.
557 4. Wade B, Keyburn. A. The true cost of necrotic enteritis. World Poultry. 2015; 31:16–7.
558 5. Liu D, Guo Y, Wang Z, Yuan J. Exogenous lysozyme influences Clostridium perfringens
559 colonization and intestinal barrier function in broiler chickens. Avian Pathol.
560 2010;39(1):17-24. Epub 2010/04/15. doi: 10.1080/03079450903447404. PubMed PMID:
561 20390532.
562 6. Marshall BM, Levy SB. Food animals and antimicrobials: impacts on human health. Clin
563 Microbiol Rev. 2011;24(4):718-33. doi: 10.1128/CMR.00002-11. PubMed PMID:
564 21976606; PubMed Central PMCID: PMCPMC3194830.
565 7. Van Immerseel F, De Buck J, Pasmans F, Huyghebaert G, Haesebrouck F, Ducatelle R.
566 Clostridium perfringens in poultry: an emerging threat for animal and public health. Avian
567 Pathol. 2004;33(6):537-49. Epub 2005/03/15. doi: 10.1080/03079450400013162. PubMed
568 PMID: 15763720.
569 8. Van Immerseel F, Rood JI, Moore RJ, Titball RW. Rethinking our understanding of the
570 pathogenesis of necrotic enteritis in chickens. Trends Microbiol. 2009;17(1):32-6. Epub
571 2008/11/04. doi: 10.1016/j.tim.2008.09.005. PubMed PMID: 18977143.
572 9. Casewell M, Friis C, Marco E, McMullin P, Phillips I. The European ban on growth-
573 promoting antibiotics and emerging consequences for human and animal health. J
44 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
574 Antimicrob Chemother. 2003;52(2):159-61. Epub 2003/07/03. doi: 10.1093/jac/dkg313.
575 PubMed PMID: 12837737.
576 10. Timbermont L, Haesebrouck F, Ducatelle R, Van Immerseel F. Necrotic enteritis in
577 broilers: an updated review on the pathogenesis. Avian Pathol. 2011;40(4):341-7. Epub
578 2011/08/05. doi: 10.1080/03079457.2011.590967. PubMed PMID: 21812711.
579 11. Gaucher ML, Quessy S, Letellier A, Arsenault J, Boulianne M. Impact of a drug-free
580 program on broiler chicken growth performances, gut health, Clostridium perfringens and
581 Campylobacter jejuni occurrences at the farm level. Poult Sci. 2015;94(8):1791-801. Epub
582 2015/06/07. doi: 10.3382/ps/pev142. PubMed PMID: 26047674.
583 12. Rehman HU, Vahjen W, Awad WA, Zentek J. Indigenous bacteria and bacterial metabolic
584 products in the gastrointestinal tract of broiler chickens. Arch Anim Nutr. 2007;61(5):319-
585 35. Epub 2007/11/23. doi: 10.1080/17450390701556817. PubMed PMID: 18030916.
586 13. Pan D, Yu Z. Intestinal microbiome of poultry and its interaction with host and diet. Gut
587 Microbes. 2014;5(1):108-19. doi: 10.4161/gmic.26945. PubMed PMID: 24256702;
588 PubMed Central PMCID: PMCPMC4049927.
589 14. Yin Y, Lei F, Zhu L, Li S, Wu Z, Zhang R, et al. Exposure of different bacterial inocula to
590 newborn chicken affects gut microbiota development and ileum gene expression. ISME J.
591 2010;4(3):367-76. doi: 10.1038/ismej.2009.128. PubMed PMID: 19956274.
592 15. Mwangi WN, Beal RK, Powers C, Wu X, Humphrey T, Watson M, et al. Regional and
593 global changes in TCRalphabeta T cell repertoires in the gut are dependent upon the
594 complexity of the enteric microflora. Dev Comp Immunol. 2010;34(4):406-17. doi:
595 10.1016/j.dci.2009.11.009. PubMed PMID: 19945480.
45 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
596 16. Crhanova M, Hradecka H, Faldynova M, Matulova M, Havlickova H, Sisak F, et al.
597 Immune response of chicken gut to natural colonization by gut microflora and to
598 Salmonella enterica serovar enteritidis infection. Infect Immun. 2011;79(7):2755-63. doi:
599 10.1128/IAI.01375-10. PubMed PMID: 21555397; PubMed Central PMCID:
600 PMCPMC3191970.
601 17. Feng Y, Gong J, Yu H, Jin Y, Zhu J, Han Y. Identification of changes in the composition
602 of ileal bacterial microbiota of broiler chickens infected with Clostridium perfringens. Vet
603 Microbiol. 2010;140(1-2):116-21. doi: 10.1016/j.vetmic.2009.07.001. PubMed PMID:
604 19647376.
605 18. Stanley D, Keyburn AL, Denman SE, Moore RJ. Changes in the caecal microflora of
606 chickens following Clostridium perfringens challenge to induce necrotic enteritis. Vet
607 Microbiol. 2012;159(1-2):155-62. Epub 2012/04/11. doi: 10.1016/j.vetmic.2012.03.032.
608 PubMed PMID: 22487456.
609 19. Stanley D, Wu SB, Rodgers N, Swick RA, Moore RJ. Differential responses of cecal
610 microbiota to fishmeal, Eimeria and Clostridium perfringens in a necrotic enteritis
611 challenge model in chickens. PLoS One. 2014;9(8):e104739. doi:
612 10.1371/journal.pone.0104739. PubMed PMID: 25167074; PubMed Central PMCID:
613 PMCPMC4148237.
614 20. Li Z, Wang W, Liu D, Guo Y. Effects of Lactobacillus acidophilus on gut microbiota
615 composition in broilers challenged with Clostridium perfringens. PLoS One.
616 2017;12(11):e0188634. doi: 10.1371/journal.pone.0188634. PubMed PMID: 29190649;
617 PubMed Central PMCID: PMCPMC5708699.
46 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
618 21. Dittmar E, Beyer P, Fischer D, Schafer V, Schoepe H, Bauer K, et al. Necrotizing
619 enterocolitis of the neonate with Clostridium perfringens: diagnosis, clinical course, and
620 role of alpha toxin. Eur J Pediatr. 2008;167(8):891-5. doi: 10.1007/s00431-007-0614-9.
621 PubMed PMID: 17952466.
622 22. Heida FH, van Zoonen AG, Hulscher JB, te Kiefte BJ, Wessels R, Kooi EM, et al. A
623 Necrotizing Enterocolitis-Associated Gut Microbiota Is Present in the Meconium: Results
624 of a Prospective Study. Clin Infect Dis. 2016;62(7):863-70. Epub 2016/01/21. doi:
625 10.1093/cid/ciw016. PubMed PMID: 26787171.
626 23. Dahiya JP, Wilkie DC, Van Kessel AG, Drew MD. Potential strategies for controlling
627 necrotic enteritis in broiler chickens in post-antibiotic era. Anim Feed Sci Technol.
628 2006;129(1-2):60-88. doi: 10.1016/j.anifeedsci.2005.12.003.
629 24. Caly DL, D'Inca R, Auclair E, Drider D. Alternatives to Antibiotics to Prevent Necrotic
630 Enteritis in Broiler Chickens: A Microbiologist's Perspective. Front Microbiol.
631 2015;6:1336. doi: 10.3389/fmicb.2015.01336. PubMed PMID: 26648920; PubMed Central
632 PMCID: PMCPMC4664614.
633 25. Hermans D, Martel A, Garmyn A, Verlinden M, Heyndrickx M, Gantois I, et al.
634 Application of medium-chain fatty acids in drinking water increases Campylobacter jejuni
635 colonization threshold in broiler chicks. Poult Sci. 2012;91(7):1733-8. doi:
636 10.3382/ps.2011-02106. PubMed PMID: 22700521.
637 26. Van Immerseel F, De Buck J, Boyen F, Bohez L, Pasmans F, Volf J, et al. Medium-chain
638 fatty acids decrease colonization and invasion through hilA suppression shortly after
639 infection of chickens with Salmonella enterica serovar Enteritidis. Appl Environ
47 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
640 Microbiol. 2004;70(6):3582-7. doi: 10.1128/AEM.70.6.3582-3587.2004. PubMed PMID:
641 15184160; PubMed Central PMCID: PMCPMC427757.
642 27. Zeiger K, Popp J, Becker A, Hankel J, Visscher C, Klein G, et al. Lauric acid as feed
643 additive - An approach to reducing Campylobacter spp. in broiler meat. PLoS One.
644 2017;12(4):e0175693. doi: 10.1371/journal.pone.0175693. PubMed PMID: 28419122;
645 PubMed Central PMCID: PMCPMC5395180.
646 28. Skrivanova E, Marounek M, Dlouha G, Kanka J. Susceptibility of Clostridium perfringens
647 to C-C fatty acids. Lett Appl Microbiol. 2005;41(1):77-81. doi: 10.1111/j.1472-
648 765X.2005.01709.x. PubMed PMID: 15960756.
649 29. Timbermont L, Lanckriet A, Dewulf J, Nollet N, Schwarzer K, Haesebrouck F, et al.
650 Control of Clostridium perfringens-induced necrotic enteritis in broilers by target-released
651 butyric acid, fatty acids and essential oils. Avian Pathol. 2010;39(2):117-21. doi:
652 10.1080/03079451003610586. PubMed PMID: 20390546.
653 30. Wu SB, Stanley D, Rodgers N, Swick RA, Moore RJ. Two necrotic enteritis predisposing
654 factors, dietary fishmeal and Eimeria infection, induce large changes in the caecal
655 microbiota of broiler chickens. Vet Microbiol. 2014;169(3-4):188-97. doi:
656 10.1016/j.vetmic.2014.01.007. PubMed PMID: 24522272.
657 31. Prescott JF, Smyth JA, Shojadoost B, Vince A. Experimental reproduction of necrotic
658 enteritis in chickens: a review. Avian Pathol. 2016;45(3):317-22. doi:
659 10.1080/03079457.2016.1141345. PubMed PMID: 26813025.
660 32. Shojadoost B, Vince AR, Prescott JF. The successful experimental induction of necrotic
661 enteritis in chickens by Clostridium perfringens: a critical review. Vet Res. 2012;43:74.
48 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
662 Epub 2012/10/30. doi: 10.1186/1297-9716-43-74. PubMed PMID: 23101966; PubMed
663 Central PMCID: PMCPMC3546943.
664 33. Prescott JF, Parreira VR, Mehdizadeh Gohari I, Lepp D, Gong J. The pathogenesis of
665 necrotic enteritis in chickens: what we know and what we need to know: a review. Avian
666 Pathol. 2016;45(3):288-94. doi: 10.1080/03079457.2016.1139688. PubMed PMID:
667 26813023.
668 34. Moore RJ. Necrotic enteritis predisposing factors in broiler chickens. Avian Pathol.
669 2016;45(3):275-81. doi: 10.1080/03079457.2016.1150587. PubMed PMID: 26926926.
670 35. Xu S, Lin Y, Zeng D, Zhou M, Zeng Y, Wang H, et al. Bacillus licheniformis normalize
671 the ileum microbiota of chickens infected with necrotic enteritis. Sci Rep. 2018;8(1):1744.
672 doi: 10.1038/s41598-018-20059-z. PubMed PMID: 29379124; PubMed Central PMCID:
673 PMCPMC5789067.
674 36. Lin Y, Xu S, Zeng D, Ni X, Zhou M, Zeng Y, et al. Disruption in the cecal microbiota of
675 chickens challenged with Clostridium perfringens and other factors was alleviated by
676 Bacillus licheniformis supplementation. PLoS One. 2017;12(8):e0182426. doi:
677 10.1371/journal.pone.0182426. PubMed PMID: 28771569; PubMed Central PMCID:
678 PMCPMC5542615.
679 37. Branton SL, Reece FN, Hagler WM, Jr. Influence of a wheat diet on mortality of broiler
680 chickens associated with necrotic enteritis. Poult Sci. 1987;66(8):1326-30. Epub
681 1987/08/01. PubMed PMID: 2891128.
682 38. Thompson DR, Parreira VR, Kulkarni RR, Prescott JF. Live attenuated vaccine-based
683 control of necrotic enteritis of broiler chickens. Vet Microbiol. 2006;113(1-2):25-34. Epub
684 2005/11/18. doi: 10.1016/j.vetmic.2005.10.015. PubMed PMID: 16289639.
49 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
685 39. Jiang Y, Kulkarni RR, Parreira VR, Prescott JF. Immunization of broiler chickens against
686 Clostridium perfringens-induced necrotic enteritis using purified recombinant
687 immunogenic proteins. Avian Dis. 2009;53(3):409-15. Epub 2009/10/24. doi:
688 10.1637/8656-021109-Reg.1. PubMed PMID: 19848081.
689 40. Yu Q, Lepp D, Mehdizadeh Gohari I, Wu T, Zhou H, Yin X, et al. The Agr-like quorum
690 sensing system is required for necrotic enteritis pathogenesis in poultry caused by
691 Clostridium perfringens. Infect Immun. 2017. Epub 2017/04/05. doi: 10.1128/iai.00975-16.
692 PubMed PMID: 28373356.
693 41. Zhou H, Lepp D, Pei Y, Liu M, Yin X, Ma R, et al. Influence of pCP1NetB ancillary genes
694 on the virulence of Clostridium perfringens poultry necrotic enteritis strain CP1. Gut
695 Pathog. 2017;9:6. doi: 10.1186/s13099-016-0152-y. PubMed PMID: 28127404; PubMed
696 Central PMCID: PMCPMC5251324.
697 42. Keyburn AL, Sheedy SA, Ford ME, Williamson MM, Awad MM, Rood JI, et al. Alpha-
698 toxin of Clostridium perfringens is not an essential virulence factor in necrotic enteritis in
699 chickens. Infect Immun. 2006;74(11):6496-500. Epub 2006/08/23. doi: 10.1128/iai.00806-
700 06. PubMed PMID: 16923791; PubMed Central PMCID: PMCPmc1695520.
701 43. Magoc T, Salzberg SL. FLASH: fast length adjustment of short reads to improve genome
702 assemblies. Bioinformatics. 2011;27(21):2957-63. doi: 10.1093/bioinformatics/btr507.
703 PubMed PMID: 21903629; PubMed Central PMCID: PMCPMC3198573.
704 44. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al.
705 QIIME allows analysis of high-throughput community sequencing data. Nat Methods.
706 2010;7(5):335-6. doi: 10.1038/nmeth.f.303. PubMed PMID: 20383131; PubMed Central
707 PMCID: PMCPMC3156573.
50 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
708 45. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R. UCHIME improves sensitivity and
709 speed of chimera detection. Bioinformatics. 2011;27(16):2194-200. doi:
710 10.1093/bioinformatics/btr381. PubMed PMID: 21700674; PubMed Central PMCID:
711 PMCPMC3150044.
712 46. Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics.
713 2010;26(19):2460-1. doi: 10.1093/bioinformatics/btq461. PubMed PMID: 20709691.
714 47. Edgar RC. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat
715 Methods. 2013;10(10):996-8. Epub 2013/08/21. doi: 10.1038/nmeth.2604. PubMed PMID:
716 23955772.
717 48. Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive Bayesian classifier for rapid assignment
718 of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol.
719 2007;73(16):5261-7. doi: 10.1128/AEM.00062-07. PubMed PMID: 17586664; PubMed
720 Central PMCID: PMCPMC1950982.
721 49. Parks DH, Tyson GW, Hugenholtz P, Beiko RG. STAMP: statistical analysis of taxonomic
722 and functional profiles. Bioinformatics. 2014;30(21):3123-4. doi:
723 10.1093/bioinformatics/btu494. PubMed PMID: 25061070; PubMed Central PMCID:
724 PMCPMC4609014.
725 50. Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, et al. Metagenomic
726 biomarker discovery and explanation. Genome Biol. 2011;12(6):R60. doi: 10.1186/gb-
727 2011-12-6-r60. PubMed PMID: 21702898; PubMed Central PMCID: PMCPMC3218848.
728 51. Antonissen G, Eeckhaut V, Van Driessche K, Onrust L, Haesebrouck F, Ducatelle R, et al.
729 Microbial shifts associated with necrotic enteritis. Avian Pathol. 2016;45(3):308-12. doi:
730 10.1080/03079457.2016.1152625. PubMed PMID: 26950294.
51 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
731 52. Sengupta R, Altermann E, Anderson RC, McNabb WC, Moughan PJ, Roy NC. The role of
732 cell surface architecture of lactobacilli in host-microbe interactions in the gastrointestinal
733 tract. Mediators Inflamm. 2013;2013:237921. Epub 2013/04/12. doi:
734 10.1155/2013/237921. PubMed PMID: 23576850; PubMed Central PMCID:
735 PMCPMC3610365.
736 53. Belenguer A, Duncan SH, Holtrop G, Anderson SE, Lobley GE, Flint HJ. Impact of pH on
737 lactate formation and utilization by human fecal microbial communities. Appl Environ
738 Microbiol. 2007;73(20):6526-33. doi: 10.1128/AEM.00508-07. PubMed PMID: 17766450;
739 PubMed Central PMCID: PMCPMC2075063.
740 54. Mora A, Herrera A, Mamani R, Lopez C, Alonso MP, Blanco JE, et al. Recent emergence
741 of clonal group O25b:K1:H4-B2-ST131 ibeA strains among Escherichia coli poultry
742 isolates, including CTX-M-9-producing strains, and comparison with clinical human
743 isolates. Appl Environ Microbiol. 2010;76(21):6991-7. doi: 10.1128/AEM.01112-10.
744 PubMed PMID: 20817805; PubMed Central PMCID: PMCPMC2976257.
745 55. Du E, Gan L, Li Z, Wang W, Liu D, Guo Y. In vitro antibacterial activity of thymol and
746 carvacrol and their effects on broiler chickens challenged with Clostridium perfringens. J
747 Anim Sci Biotechnol. 2015;6:58. doi: 10.1186/s40104-015-0055-7. PubMed PMID:
748 26705471; PubMed Central PMCID: PMCPMC4690362.
749 56. Du E, Wang W, Gan L, Li Z, Guo S, Guo Y. Effects of thymol and carvacrol
750 supplementation on intestinal integrity and immune responses of broiler chickens
751 challenged with Clostridium perfringens. J Anim Sci Biotechnol. 2016;7:19. doi:
752 10.1186/s40104-016-0079-7. PubMed PMID: 27006768; PubMed Central PMCID:
753 PMCPMC4802587.
52 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
754 57. Fusco V, Quero GM, Cho GS, Kabisch J, Meske D, Neve H, et al. The genus Weissella:
755 taxonomy, ecology and biotechnological potential. Front Microbiol. 2015;6:155. doi:
756 10.3389/fmicb.2015.00155. PubMed PMID: 25852652; PubMed Central PMCID:
757 PMCPMC4362408.
758 58. Collins MD, Lawson PA, Willems A, Cordoba JJ, Fernandez-Garayzabal J, Garcia P, et al.
759 The phylogeny of the genus Clostridium: proposal of five new genera and eleven new
760 species combinations. Int J Syst Bacteriol. 1994;44(4):812-26. Epub 1994/10/01. doi:
761 10.1099/00207713-44-4-812. PubMed PMID: 7981107.
762 59. Stackebrandt E, Kramer I, Swiderski J, Hippe H. Phylogenetic basis for a taxonomic
763 dissection of the genus Clostridium. FEMS Immunol Med Microbiol. 1999;24(3):253-8.
764 Epub 1999/07/09. PubMed PMID: 10397308.
765 60. Rajilic-Stojanovic M, de Vos WM. The first 1000 cultured species of the human
766 gastrointestinal microbiota. FEMS Microbiol Rev. 2014;38(5):996-1047. doi:
767 10.1111/1574-6976.12075. PubMed PMID: 24861948; PubMed Central PMCID:
768 PMCPMC4262072.
769 61. Lakshminarayanan B, Harris HM, Coakley M, O'Sullivan O, Stanton C, Pruteanu M, et al.
770 Prevalence and characterization of Clostridium perfringens from the faecal microbiota of
771 elderly Irish subjects. J Med Microbiol. 2013;62(Pt 3):457-66. Epub 2012/12/12. doi:
772 10.1099/jmm.0.052258-0. PubMed PMID: 23222860.
773 62. Williams RB, Marshall RN, La Ragione RM, Catchpole J. A new method for the
774 experimental production of necrotic enteritis and its use for studies on the relationships
775 between necrotic enteritis, coccidiosis and anticoccidial vaccination of chickens. Parasitol
53 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
776 Res. 2003;90(1):19-26. Epub 2003/05/14. doi: 10.1007/s00436-002-0803-4. PubMed
777 PMID: 12743800.
778 63. Williams RB. Intercurrent coccidiosis and necrotic enteritis of chickens: rational,
779 integrated disease management by maintenance of gut integrity. Avian Pathol.
780 2005;34(3):159-80. Epub 2005/09/30. doi: 10.1080/03079450500112195. PubMed PMID:
781 16191699.
782 64. Li C, Lillehoj HS, Gadde UD, Ritter D, Oh S. Characterization of Clostridium perfringens
783 Strains Isolated from Healthy and Necrotic Enteritis-Afflicted Broiler Chickens. Avian
784 Dis. 2017;61(2):178-85. Epub 2017/07/01. doi: 10.1637/11507-093016-Reg.1. PubMed
785 PMID: 28665720.
786 65. Yan W, Sun C, Yuan J, Yang N. Gut metagenomic analysis reveals prominent roles of
787 Lactobacillus and cecal microbiota in chicken feed efficiency. Sci Rep. 2017;7:45308. doi:
788 10.1038/srep45308. PubMed PMID: 28349946.
789 66. Wohlgemuth S, Keller S, Kertscher R, Stadion M, Haller D, Kisling S, et al. Intestinal
790 steroid profiles and microbiota composition in colitic mice. Gut Microbes. 2011;2(3):159-
791 66. doi: 10.4161/gmic.2.3.16104. PubMed PMID: 21869607.
792 67. Stanley D, Denman SE, Hughes RJ, Geier MS, Crowley TM, Chen H, et al. Intestinal
793 microbiota associated with differential feed conversion efficiency in chickens. Appl
794 Microbiol Biotechnol. 2012;96(5):1361-9. Epub 2012/01/18. doi: 10.1007/s00253-011-
795 3847-5. PubMed PMID: 22249719.
796 68. Xiao Y, Xiang Y, Zhou W, Chen J, Li K, Yang H. Microbial community mapping in
797 intestinal tract of broiler chicken. Poult Sci. 2017;96(5):1387-93. doi: 10.3382/ps/pew372.
798 PubMed PMID: 28339527.
54 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
799 69. Yeoman CJ, Chia N, Jeraldo P, Sipos M, Goldenfeld ND, White BA. The microbiome of
800 the chicken gastrointestinal tract. Anim Health Res Rev. 2012;13(1):89-99. doi:
801 10.1017/S1466252312000138. PubMed PMID: 22853945.
802 70. Dierick N, Michiels J, Van Nevel C. Effect of medium chain fatty acids and benzoic acid,
803 as alternatives for antibiotics, on growth and some gut parameters in piglets. Commun
804 Agric Appl Biol Sci. 2004;69(2):187-90. Epub 2004/11/25. PubMed PMID: 15560218.
805 71. Bertevello PL, De Nardi L, Torrinhas RS, Logullo AF, Waitzberg DL. Partial replacement
806 of omega-6 fatty acids with medium-chain triglycerides, but not olive oil, improves colon
807 cytokine response and damage in experimental colitis. JPEN J Parenter Enteral Nutr.
808 2012;36(4):442-8. Epub 2012/01/25. doi: 10.1177/0148607111421788. PubMed PMID:
809 22269895.
810 72. Zentek J, Buchheit-Renko S, Manner K, Pieper R, Vahjen W. Intestinal concentrations of
811 free and encapsulated dietary medium-chain fatty acids and effects on gastric microbial
812 ecology and bacterial metabolic products in the digestive tract of piglets. Arch Anim Nutr.
813 2012;66(1):14-26. Epub 2012/03/09. PubMed PMID: 22397093.
814 73. Desbois AP, Smith VJ. Antibacterial free fatty acids: activities, mechanisms of action and
815 biotechnological potential. Appl Microbiol Biotechnol. 2010;85(6):1629-42. doi:
816 10.1007/s00253-009-2355-3. PubMed PMID: 19956944.
817 74. Altieri C, Bevilacqua A, Cardillo D, Sinigaglia M. Effectiveness of fatty acids and their
818 monoglycerides against gram-negative pathogens. Int J Food Sci Tech. 2009;44(2):359-66.
819 doi: 10.1111/j.1365-2621.2008.01744.x.
55 bioRxiv preprint doi: https://doi.org/10.1101/434449; this version posted October 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license.
820 75. Bergsson G, Arnfinnsson J, Steingrimsson O, Thormar H. Killing of Gram-positive cocci
821 by fatty acids and monoglycerides. APMIS. 2001;109(10):670-8. Epub 2002/03/14.
822 PubMed PMID: 11890570.
823 76. Mathis G, T. P. van Dam J, Corujo Fernández A, L. Hofacre C. Effect of an organic acids
824 and medium-chain fatty acids containing product in feed on the course of artificial Necrotic
825 Enteritis infection in broiler chickens2018.
826 77. Zentek J, Buchheit-Renko S, Ferrara F, Vahjen W, Van Kessel AG, Pieper R. Nutritional
827 and physiological role of medium-chain triglycerides and medium-chain fatty acids in
828 piglets. Anim Health Res Rev. 2011;12(1):83-93. doi: 10.1017/S1466252311000089.
829 PubMed PMID: 21676342.
830
831 Supporting information
832 Figure S1. Rarefaction curve by groups.
833 Figure S2. Comparison of genera abundance between jejunal and cecal microbiota by STAMP
834 with Welch's t-test.
835 Figure S3. Rank abundance curve by groups.
836 Figure S4. Comparison of genera (A) and species (B) abundance between AJ and EJ, BJ and EJ,
837 and CJ and EJ groups by STAMP with Welch's t-test.
56